Treatment and prevention of tissue damage

ABSTRACT

A method for the treatment or prevention of tissue damage in a subject having an inflammatory and/or tissue damaging condition, which comprises administering to the subject an effective amount of a compound capable of inhibiting the binding of C-reactive protein (CRP) to an autologous or extrinsic ligand.

The present invention relates to a method for the treatment orprevention of tissue damage in a subject, especially a human subjecthaving an inflammatory and/or tissue damaging condition. Compounds areprovided for the treatment or prevention of the tissue damage, as wellas methods for selecting and producing such compounds.

C-reactive protein (CRP) is a normal plasma protein of the pentraxinprotein family, the other member of which is the very closely similarmolecule, serum amyloid P component (SAP)¹. CRP is the classical acutephase protein, the circulating concentration of which increasesdramatically in response to most forms of inflammation, tissue injuryand infection, and the value attained in most conditions correlatesclosely with the extent and activity of disease CRP is a calciumdependent ligand binding protein, the ligand which it binds with highestaffinity being phosphocholine residues², but it also binds a variety ofother ligands. It binds many of its ligands with high avidity. Knownligands for CRP include both autologous and extrinsic structures.Autologous ligands include native^(4,5) and modified plasmalipoproteins, damaged cell membranes⁶, a number of differentphospholipids and related compounds⁷, and small nuclearribonucleoprotein particles^(8,9). Extrinsic ligands include manyglycan, phospholipid and other components of micro-organisms, such ascapsular and somatic components of bacteria, fungi and parasites, aswell as plant products¹⁰⁻¹⁵. When CRP has bound to its ligands itbecomes capable of activating complement by the classical pathway viaClq¹⁶⁻¹⁹ and achieving activation and fixation of C3, the main adhesionmolecule of the complement system^(20,21), as well as engagement of theterminal lytic phase, C5-C9²².

Whilst very early clinical work²³ suggested that CRP might possiblycontribute to inflammation, and subsequent experimental animal studieswere interpreted as showing a pro-inflammatory role for CRP, there hashitherto been no direct evidence of any involvement of CRP in processesof inflammation and tissue damage. There are a few reports of CRPdeposition in inflammatory and necrotic tissue lesions, and ofassociation between CRP and complement activation²⁴⁻³⁰. However none ofthese studies shows directly that CRP is responsible for tissue damage,and the only study of real time CRP deposition in human tissues inliving patients showed that it occurred only in trace amounts, if atall³¹. Indeed the published work that directly examines the role of CRPin experimental models of disease indicates that CRP may have ananti-inflammatory role that down-regulates infiltration of inflammatorycells and reduces tissue damage^(32,33). This would be consistent withthe finding that complexed CRP is relatively inefficient at generatingthe terminal phase of complement activation and that involvement of CRPdown-regulates other potentially inflammatory aspects of complementactivation^(34,35). Very recent work in different models involvinghandling of apoptotic cells also indicates that CRP hasanti-inflammatory properties³⁶. There is thus certainly no consensusabout the role of CRP in vivo and the predominant view is that it may beanti-inflammatory. In general the association of increased CRPproduction with disease conditions has hitherto been interpreted on thebasis that CRP production reflects the severity of the underlyingdisease and/or the presence of intercurrent complications.

Atherosclerosis is extremely prevalent in developed countries and itsmajor complications of myocardial infarction and stroke together accountfor about one third of all deaths. Although there have been advances inunderstanding of some aspects of pathogenesis and in prophylactic andpost-event salvage treatments, the personal, social and economic burdenof these conditions remains enormous. Similarly, chronic inflammatorydiseases of unknown aetiology are common, debilitating, expensive andoften dangerous to treat symptomatically, as well as being incurable andoften shortening life expectancy. For example, rheumatoid arthritisaffects about 4% of the population over the age of 50 years and, as wellas being painful and causing severe disability, it is associated withsignificant premature mortality. The cancer burden is very heavy,accounting for about one third of all deaths in developed countries, andthe severity and importance of infectious disease throughout the worldis evident. There is a pressing need for new drugs to reduce severityand to prolong survival in all these different conditions.

Accordingly, in a first aspect, the present invention provides a methodfor the treatment or prevention of tissue damage in a subject having aninflammatory and/or tissue damaging condition, which comprisesadministering to the subject an effective amount of a compound capableof inhibiting the binding of C-reactive protein (CRP) to an autologousor extrinsic ligand.

As reported herein, it has been found for the first time that CRP doesplay a direct pathogenetic role in a disease condition, specifically byenhancing the extent of myocardial damage produced by ischemic injury.This pathogenetic role can be treated or prevented by the use of a drugcapable of inhibiting the binding of CRP to its target ligand in vivo.Without wishing to be bound by theory and as described in further detailherein, it is thought that the inhibition of binding of CRP to itstarget ligand in vivo would prevent CRP from activating complement andthereby reduce or eliminate the deleterious effects of CRP mediatedcomplement activation now thought responsible for tissue damage in theconditions to be treated according to the present invention.

In one embodiment according to the invention, the inflammatory and/ortissue damaging condition comprises atherosclerosis.

Whilst CRP is produced in large amounts in response to most forms oftissue injury, inflammation and infection, its circulating concentrationis extremely low in normal healthy subjects and in most individuals inthe general population^(37,38). Until recently these low levels were notconsidered to be of any clinical significance and the generallyavailable assays for CRP were designed only to detect and measurecirculating CRP when the concentration exceeded 5 or even 10 mg/l,representing the 90-99^(th) centile of the range found in healthysubjects. However a large body of work has accumulated since ouroriginal discoveries, starting in 1994³⁹, that shows convincingly thateven within the reference range for CRP, among values previouslyconsidered to be “normal”, increased production of CRP is verysignificantly associated with atherothrombotic events, includingmyocardial infarction, stroke and progression of vascular disease⁴⁰⁻⁴⁸.

The mechanisms underlying the association between even modestlyincreased CRP production and development, progression and complicationsof atherosclerosis, are not known. However it is likely to be highlyrelevant that atherosclerosis is known to be an inflammatory condition,and that CRP and activated complement are co-deposited in virtually allatheromatous plaques^(29,49). Furthermore, CRP selectively binds to lowdensity lipoprotein (LDL), the major lipoprotein that accumulates in thearterial lesions of atherosclerosis^(4,5), and binding of CRP to“modified”, that is partly degraded, LDL such as is found in theplaques, potently activates complement⁵⁰. Finally there is evidence thatCRP can stimulate macrophages, which are the most abundant cellsinfiltrating atheromatous plaques, to produce tissue factor (TF)⁵¹. TFis the initiator of blood coagulation responsible for initiation of thethrombus formation on ruptured plaques that actually occludesatherosclerotic arteries and precipitates myocardial infarction orstroke. CRP may thus directly contribute to the pathogenesis,progression and clinically significant complications of atherosclerosis.

Once myocardial infarction has occurred, all patients mount a majoracute phase response of CRP and the peak value attained is verysignificantly prognostic of outcome, that is complications and death,over the ensuing days, weeks and months⁵²⁻⁵⁸. Given the universalco-deposition of CRP and activated complement within the infarct itself,this strongly suggests that CRP contributes importantly to the extentand severity of the ischaemic pathology^(28,59,60). CRP values, andparticularly cumulative production of CRP over time, are also verysignificantly predictive of progression, severity and complications ofchronic inflammatory diseases of unknown aetiology, such as rheumatoidarthritis⁶¹ and Crohn's disease, of acute and chronic bacterial, viral,fungal and parasitic infections, of ischaemic and necrotic diseases suchas acute pancreatitis, and of many forms of cancer (reviewed in^(1,2)).Even in the context of elective surgery, pre-operative CRP values andpost-operative CRP production predict complications and outcome⁶². Ourobservations in atherosclerosis, and especially in the rat model ofmyocardial infarction exacerbated by human CRP, now indicate that CRPmay actually be actively contributing to disease severity in all thesedifferent conditions

In a further embodiment, the inflammatory and/or tissue damagingcondition is selected from an infection, an allergic complication ofinfection, an inflammatory disease, ischemic or other necrosis,traumatic tissue damage and malignant neoplasia.

For example, where the condition is an infection, this may be anon-protozoal infection such as a bacterial or viral infection. Wherethe condition is an allergic complication of infection, this may beselected from rheumatic fever, glomerulonephritis and erythema nodosumleprosum. Where the condition is an inflammatory disease, this may beselected from rheumatoid arthritis, juvenile chronic (rheumatoid)arthritis, ankylosing spondylitis, psoriatic arthritis, systemicvasculitis, polymyalgia rheumatica, Reiter's disease, Crohn's diseaseand familial Mediterranean fever. Where the condition involves ischaemicor other necrosis selected from myocardial infarction, tumourembolization and acute pancreatitis. Where the condition is traumatic,this may be selected from acute or elective surgery, burns, chemical orphysical injury, and fractures. Where the condition is malignantneoplasia, this may be selected from lymphoma, Hodgkin's disease,carcinoma and sarcoma.

According to the present invention, drugs that either inhibit thebinding of CRP to its ligands in vivo, and/or that reduce itsavailability for such binding in vivo, will block the contribution ofCRP to pathogenesis of disease and will thereby reduce extent andseverity of disease, reducing symptoms and prolonging survival. Thepresent invention provides for identification and testing of compoundswith such effects, for the preparation of a composition for theprevention and/or treatment of atherosclerosis and its complications,including myocardial infarction, stroke and peripheral vascular disease,acute and chronic inflammatory diseases of known and unknown aetiology,acute and chronic infections of all types, traumatic injuries includingburns, acute and elective surgery, malignant neoplasia of all types, andall disease conditions associated with increased CRP production.

Accordingly, in a further aspect, the present invention provides amethod for selecting a pharmaceutical compound for treating orpreventing tissue damage in a subject having an inflammatory and/ortissue damaging condition, which comprises contacting C-reactive protein(CRP) with a ligand thereof under conditions to permit CRP ligandbinding, in the presence of a test compound; and selecting the testcompound as the pharmaceutical compound If the test compound inhibitsbinding of CRP to the ligand.

The present invention further provides a process for the production of apharmaceutical agent. This process comprises (1) identifying apharmaceutical compound by selecting the compound as described above;and (2) producing a pharmaceutical agent by providing a pharmaceuticalcompound or a pharmaceutical-acceptable derivative thereof.

The present invention is therefore concerned with a method for selectinga pharmaceutical compound which includes testing for CRP ligand bindingin the presence of a test compound. Any test compound which inhibitsbinding of SAP to the ligand is selected as a potential pharmaceutical.For example, the test compound may be selected in the sense that it isidentified and can then be produced on a larger scale by chemical orbiochemical synthesis or may be physically selected for directformulation as a pharmaceutical. In accordance with the process forproduction of the pharmaceutical agent, the test compound may beformulated for pharmaceutical use or may be derivatised or chemicallymodified to produce a pharmaceutically-acceptable derivative thereof.Such derivatisation may simply be required to incorporate new functionalgroups or alter existing functional groups to make the agent easier toformulate, for example by altering the solubility of the compound.Derivatisation of this nature may be used to decrease the toxicity ofthe compound, to alter the stability of the compound or even to modifythe pharmacological activity thereof. Any such derivatised or modifiedcompound may need to be retested according to the method of the presentinvention.

In the step of contacting CRP with the ligand, the conditions must besufficient to permit CRP ligand binding in the absence of the testcompound. In this way, where CRP ligand binding does not occur in thepresence of the test compound, or occurs to a smaller extent thanexpected, this effect can be attributed to the test compound. It shouldbe noted here that inhibition of binding should be broadly construed andis not limited to any particular mechanism; any reduction of the extentof binding constitutes inhibition of binding according to the presentinvention. Inhibition of binding is generally measured with reference toa control value (maximum binding in absence of test compound) and it ispreferred that the IC₅₀ be low micromolar or less, more preferablynanomolar or less. Contacting takes place under conditions which includesufficient free calcium ions to permit the specific calcium dependentbinding of CRP. A preferred buffer for the contacting is physiologicalbuffered saline. CRP may be provided in pure or isolated form orincorporated in whole serum.

The order in which the CRP, ligand and test compound are contactedtogether is not critical. All three components can be mixed atessentially the same time or two of the three components can be mixedand perhaps pre-incubated before addition of the third component.Contacting generally takes place under conditions in which at least oneof the components is in the liquid phase. It is convenient, however, foreither the CRP or the ligand to form part of a solid phase so that, inthe testing procedure, phase separation can be used as a technique toseparate bound species from unbound species to facilitate testing forthe extent of CRP ligand binding.

Accordingly, it is preferred that a first component comprising one ofCRP or the ligand is present as part of a solid phase, which iscontacted with a second component comprising the other as part of aliquid phase. The step of testing for CRP ligand binding may thencomprise detecting binding of the second component to the solid phase.Detecting binding of the second component to the solid phase may beeffected either by detecting the presence of the second component on thesolid phase or by determining the amount of second component unbound tothe solid phase and deducing from the amount of second componentoriginally applied to the solid phase the amount actually binding to thesolid phase.

According to this embodiment, the solid phase preferably comprises thefirst component attached to a solid support, which solid support maycomprise a particulate support or a solid surface. In a convenientembodiment, the solid surface comprises an interior surface of thecontainer such as a microtitre plate well.

Conveniently, the step of testing for CRP ligand binding furthercomprises washing the solid phase to remove unbound material.

The second component may be labelled with a detectable label such as aradiolabel, a fluorochrome or an enzyme, as discussed herein.Alternatively, the binding of the second component to the solid phasemay be detected immunologically either by antibody binding to the secondcomponent as bound to the solid phase or by quantitative immunologicaldetermination of the amount of second component not bound to the solidphase.

The present invention provides in vitro spot tests, low throughput, andhigh throughput screening procedures for detecting compounds with thecapacity to inhibit binding of CRP, from man or other animals, to anyand all of its known biological and chemical ligands. These methods aresuitable for screening compound libraries of natural compounds oforganic, inorganic and biological origin, as well as chemical librariescreated by conventional synthesis or any form of combinatorialchemistry. They are also suitable for analysis of the mechanism ofinhibition of CRP binding, and for evaluation of potency of inhibitionduring chemical and medicinal chemistry development of potential oractual pharmaceutical products from lead compounds identified byscreening or spot testing. The present invention also comprises in vivomethods for testing effects and potency of CRP-inhibitory compounds onCRP binding, plasma turnover and catabolism in man and experimentalanimals, and on experimental models of disease that are exacerbated byhuman CRP.

Accordingly, in a further aspect, the present invention provides amethod for selecting a pharmaceutical compound for treating orpreventing tissue damage from a plurality of test compounds whichcomprises providing an array of reaction zones and a plurality of testcompounds, and selecting the pharmaceutical compound by performing theabove method of selecting the compound in each reaction zone.

Suitable compounds may be bound by CRP and thereby block the site ofinteraction between CRP and the target ligand, or they may bind to CRPto alter its structure thereby to inhibit or prevent binding to theligand, or they may bind to the ligand and thereby mask it and preventits recognition by CRP. The CRP molecule has a specificcalcium-dependent binding site through which it binds to its ligands,and we have characterised this at atomic resolution by X-raycrystallography A major class of compounds that can be identified usingthe present invention comprises substances that are bound by thecalcium-dependent ligand binding site of CRP so as to interfere withbinding of CRP to its ligands. Our knowledge of the 3D structure of thebinding site and the mechanism by which it binds its ligand⁶⁴ providesfor molecular design of model compounds for the purpose of the presentinvention, using for example the methods described in WO95/05394 andmore recent refinements of structure-based drug design that havesubsequently been developed. The same processes can be applied toimprove the properties of lead compounds discovered during screening oflibraries and other collections of candidate compounds. In particular,the hydrophobic cleft adjacent to the site in which phosphocholine, thehighest affinity natural ligand for CRP, is bound, is a suitable targetfor drug design⁶⁴.

The present invention also provides for detection and study of compoundsthat inhibit CRP binding by the other mechanisms listed above.Furthermore, since the goal of treatment and/or prevention of diseaseusing inhibitors of CRP binding is to prevent CRP from binding to itspathophysiologically relevant ligands in vivo, another class ofcompounds, which may or may not overlap with those described above,comprises substances that modify the availability of CRP in vivo, forexample, by accelerating its clearance from the plasma so that thecirculating concentration of CRP is greatly reduced.

One general class of compound suitable for use in the method of presentinvention has general formula (I):

wherein X is H or an organic substituent group, and Y is N substitutedto form ammonium.

X is typically H or a hydrocarbyl substituent optionally containing oneor more of O, S or N. The hydrocarbyl substituent may be an alkyl groupand is preferably unbranched. The hydrocarbyl substituent may have up to20 carbon atoms and is advantageously lipophilic. For example, X ispreferably H or C₁ to C₂₀, more preferably C₁₂ to C₂₀ alkyl. Aparticularly preferred embodiment is where X is a hexadecyl group.

It is preferred that Y is N—R₃ in which each R is independently selectedfrom C₁ to C₅ alkyl. According to this embodiment R is advantageouslymethyl. Alternatively, two of the R groups may denote a C₂ to C₅alkylene, optionally containing O, S or N.

Particularly preferred compounds include phosphocholine (where X═H andeach R═CH₃) and hexadecyl phosphocholine (where X═C₁₆ alkyl and eachR═CH₃).

The above class of chemical compounds, including methods of manufacture,are described in U.S. Pat. No. 5,990,915, which is directed to the useof these compounds in the treatment of leishmaniasis.

The invention will now be described in further detail, by way of exampleonly, with reference to the following Examples and Experiments, and theaccompanying drawings, in which:

FIG. 1 shows a graph of serum concentration against time demonstratingplasma clearance of human CRP and SAP after injection into rats; and

FIG. 2 shows the results of immunohistochemical staining of ratmyocardial infarcts.

EXAMPLES Methods for Showing Binding of CRP to Autologous and ExtrinsicLigands

In order to identify compounds that inhibit binding of CRP to ligandsthat may be relevant to the pathogenetic role of CRP, it is firstnecessary to have methods for showing such binding.

1] Binding of CRP to Autologous and Extrinsic Ligands.

For the purposes of this invention, the binding of CRP to variousligands can be demonstrated directly by allowing CRP, provided either bywhole acute phase human or animal serum, or in isolated purified form,to contact ligands of human, animal, microbial or parasitic origin, orligands derived by chemical synthesis. Examples of such ligands includenative and modified forms of natural or synthetic lipids, phospholipids,glycolipids, glycans, proteoglycans, lipoproteins, proteins, peptides,nucleic acids, oligonucleotides, mononucleotides, plasma and othercellular membrane constituents, small nuclear ribonucleoproteinparticles and other cell nuclear components, phosphocholine,phosphoethanolamine and related compounds, phosphate, sulphate,carboxylate and other polyanions. Contact must take place inphysiological buffered saline containing sufficient free calcium ions(about 2 mmol/l). The potential ligands, can be in particulate form orimmobilized on particles, such as agarose, acrylamide, polystyrene,latex, cellulose, or other beads, or on membranes, filters, or plasticor other solid surfaces such as microtitre plates or individual tubes.Immobilization may be by direct non-specific adherence of the ligand tothe particles or surfaces, or by covalent attachment via amino, hydroxy,or other chemical groups on the ligand molecules being coupled directlyor via spacer linkers to the solid phase material. After contacting thesolid or immobilised ligands, CRP that has not bound is washed away withthe same buffer in which binding took place, and the presence of CRPbound to the ligands is detected and quantified. Washing involves phaseseparation, such as centrifugation of solid particles, or immersion,flow through or flow over of solid surfaces such as membranes, filters,and plastic or other surfaces. Bound CRP may be detected directly if thesource of CRP contains CRP that has been labelled with a detectablemarker. Such markers include gamma-emitting isotopes such as ¹²⁵I or¹³¹I for detection in a gamma counter; beta-emitting isotopes such as¹⁴C or ³H for detection in a beta or scintillation counter;fluorochromes for detection in a fluorimeter, flow cytometer, orfluorescence activated cell sorter; enzymes such as peroxidase oralkaline phosphatase for detection by their specific catalytic activity.In all of these cases it is essential to demonstrate that the process ofdirectly labelling the CRP does not alter its physiological bindingproperties. This is done, for example, by comparing the binding oflabelled and unlabelled CRP to an immobilised solid phase ligand, suchas phosphoethanolamine or phosphocholine attached using a carbodiimideto carboxyhexyl-Sepharose™. Binding of CRP can also be demonstrateddirectly by immunochemical assay showing depletion of CRP from theoffered source of CRP, and recovery of the bound CRP when, after firstwashing with calcium containing buffer, the ligand material is elutedwith buffer containing EDTA to chelate calcium ions. Alternatively,bound CRP may be detected indirectly, using antibodies raised inrabbits, sheep, goats, rats, mice, guinea pigs or other animals,specific for the CRP of the species being tested. For this purpose theanti-CRP antibodies may themselves be directly labelled with aradioactive isotope, enzyme, fluorochrome or other detectable marker, orthe binding of anti-CRP antibodies to bound CRP may be detected using asecond antibody directed against the immunoglobulin of the species ofthe primary anti-CRP reagent. In addition to detection and counting ininstruments appropriate for the marker used, binding of CRP tomicro-organisms or their components may be visualised directly orindirectly using light, fluorescence or electron microscopy. Enzymelabelled CRP or anti-CRP antibodies can be used for light or electronmicroscopy, fluorochrome labelled reagents for fluorescence microscopy,and gold (or other electron dense particle) labelling for electronmicroscopy.

2] Binding of Autologous or Extrinsic Ligands by CRP.

An alternative approach to demonstration of ligand binding by CRP is toimmobilize the CRP on a solid phase and then allow it to bind ligandsthat are either directly labelled or that can be detected, for exampleusing specific antibodies directed against these ligands. Thus isolatedpurified CRP from man or other animals can be immobilized on beads,particles, membranes, filters, or plastic or other solid surfaces, bydirect non-specific adherence or by covalent coupling, or by trappingwith specific anti-CRP antibodies immobilized on the solid phase. Usingthe conditions specified in 1] above, potential ligands can then becontacted to the immobilized CRP and allowed to be bound by it.

Inhibition of CRP Binding to Microbial and Other Ligands

Any of the methods set out in 1] and 2] above for showing ligand bindingby CRP can be used to test the capacity of compounds to inhibit suchbinding. However the speed and ease of use of the different techniquesvary greatly, as well as their suitability for different purposes. Thusfor screening large numbers of compounds, high throughput methods, suchas those based on microtitre plates, are essential. A typical method ofthis type involves having ligand immobilized on the plates, and offeringto each well an amount of radiolabelled CRP under conditions such thatabout 40% of it is bound. Compounds to be tested are added to the wellsand preincubated in them before addition of the labelled CRP, and theeffect of their presence on subsequent binding of CRP is monitored. Inanother configuration, the compounds to be tested are preincubated withthe labelled CRP before the mixture is added to the plates. The reverseconfiguration, in which the CRP is immobilized, is also informative.Here the test compounds are preincubated with the immobilized CRP beforethe detectable ligand is added. These different approaches enabledetection of compounds that block ligand binding by CRP by differentmechanisms, and help to distinguish between those that are themselvesspecific ligands for CRP, those that affect the CRP molecule in otherways, and those that interact with the ligand to prevent its recognitionby CRP. The calcium dependent ligand binding site of CRP is very wellcharacterised and compounds bound by it that block binding of CRP tomodel ligands are virtually certain to also block binding of CRP topathologically and therapeutically relevant ligands. However for thepurpose of this invention, screening and subsequent development andtesting involves parallel use of assays including pathophysiological andmodel ligands. Furthermore there may be compounds that differ in therange of interactions between CRP and pathophysiological ligands thatthey inhibit. Such compounds will be of greater therapeutic value insome diseases than others, and this can only be established by testingin in vivo animal models and eventually in clinical situations.

Direct Interaction Between CRP and Test Compounds

For the purposes of the present invention, another method to identifycompounds that are bound by, or themselves bind to CRP, either in acalcium dependent fashion or independently of calcium, is the use ofsurface plasmon resonance (SPR). Purified CRP immobilized within an SPRinstrument gives a quantifiable signal when it is exposed to anothermolecule that forms a complex with the CRP, and this is distinct fromthe absence of such a signal if no complex is formed. This techniqueallows compounds to be screened for their capacity to interact with CRP,and does not depend on any specific mode of interaction with CRP, inparticular involving the calcium dependent ligand binding site of CRP,so it detects molecules that might not be found in test systems thatrequire calcium dependent CRP binding.

Effects on CRP in Vivo of Compounds that Block CRP-Ligand Binding InVitro or Interact with CRP in Other Ways.

For the purpose of the present invention, compounds that block bindingof CRP to its ligands, or that interact with CRP in other ways, aretested in vivo in mice and other experimental animals for their effectson plasma CRP concentrations and the turnover and catabolism of CRP.Having established by the administration of graded doses that thecompounds are not intrinsically toxic, they are given in a range ofdoses to inbred mice of the C57BL/6, CBA, Balb/c, or other inbred oroutbred strains. Serum is taken at regular intervals for immunochemicalassay of CRP. Trace radiolabelled human CRP, or in separate experimentsCRP from other species, is injected intravenously at different times inrelation to the drug dosage, and both whole body counting and bloodsampling is performed to monitor the plasma half life and whole bodyclearance of CRP. Plasma CRP concentration can also be monitored byspecific immunoassay, in which case native unlabelled CRP tracer can beused. In addition the tracer CRP is preincubated with different amountsof the compounds being tested, and then injected into mice not receivingany drug, in order to test the effect drug binding has on CRP clearance.The effect of compounds on CRP in vivo is also tested in mice transgenicfor human or rabbit CRP, and expressing the corresponding protein underthe control of its own or another promoter or control sequence. Bloodsamples are taken at intervals before and after administration ofdifferent drug doses, whilst increased CRP production is induced eitherby an inflammatory stimulus or however the relevant promoter istriggered. According to the present invention, compounds that accelerateCRP clearance in vivo and/or lower plasma CRP concentration, therebyreducing in vivo availability of CRP, will be of therapeutic value.

Effects of CRP Blocking and/or Binding Compounds on Pathogenicity of CRPIn Vivo.

For the purpose of the present invention, compounds that block bindingof CRP to its ligands, or that interact with CRP in other ways, aretested in vivo in mice and other experimental animals for their effectson models of disease in which human CRP exacerbates pathogenesis. Forexample, administration of human CRP to rats in which the coronaryartery is ligated to induce myocardial infarction, markedly increasesmorbidity, mortality and the infarct size⁶⁵. CRP interactive compoundsfor the purpose of the present invention, when given at the same time asthe human CRP, abrogate this enhancement of pathology and protectagainst its clinical consequences. The same experimental test, withregard to myocardial infarction induced by coronary artery ligation, isconducted in wild type mice, to which human CRP is administered, and inhuman CRP transgenic mice. Compounds are also tested in correspondingrat and mouse models of acute and chronic infection, inflammation,trauma and other pathologies that are exacerbated by the presence ofhuman CRP.

Atherosclerosis is induced in wild type C57BL/6 and other mouse strainsby provision of a high fat diet, and takes place in an accelerated andenhanced form in mice with targeted deletion of the gene for the LDLreceptor or for apolipoprotein E. In all these models, development andseverity of atherosclerosis is enhanced by intercurrent systemicinflammation, and probably also by passive administration or transgenicproduction of human CRP. CRP interactive compounds for the purpose ofthe present invention are therefore tested in these models to show thatthey abrogate the enhanced atherosclerosis engendered by human CRP.

Compounds that block CRP binding to pathophysiologically relevantligands in experimental animals in vivo and/or reduce availability ofCRP in vivo, and that have then undergone formal toxicity testing andfound to be acceptable for administration in man, are evaluated fortheir effects on plasma CRP concentration, half life, turnover andcatabolism in human subjects. Isolated human CRP is trace radiolabelledwith ¹²⁵I and injected intravenously, followed by plasma and urineturnover studies, as described elsewhere³¹, and total plasma CRPconcentration is also monitored immunochemically.

Inhibition of Binding and/or Availability of C-Reactive Protein (CRP) InVitro as a Therapeutic Modality in Disease.

The present invention relates to methods for identification of chemicalcompounds that inhibit binding, and/or the availability for binding invivo, of the plasma protein, C-reactive protein (CRP), to molecularligands of autologous or extrinsic origin. Any such compounds or theirderivatives that are acceptable for pharmaceutical use, being suitablefor the treatment and prevention of atherosclerosis, atherothromboticevents, including myocardial infarction, stroke and peripheral vasculardisease, and all types of inflammatory or tissue damaging diseases ofknown or unknown cause which are associated with increased production ofCRP.

Screening for Inhibitors of Binding of ¹²⁵I Radiolabelled CRP to EnzymeModified Low Density Lipoprotein Immobilized in Microtitre Plates.Introduction

The most specific known ligand of CRP, that is bound by it with highestaffinity, is phosphocholine. However, likely critical ligands ofimportance in vivo in the pathogenesis of atherothrombosis, are modifiedforms of low density lipoprotein. The goal of therapy with a drugcapable of inhibiting pathogenic effects of CRP in vivo is to preventbinding of CRP to such ligands. An assay to detect such compounds wastherefore established and used to test hexadecyl phosphocholine, thecandidate compound of the present invention.

Materials and Methods

Low density lipoprotein isolated from normal human serum by the standardultracentrifugation method was subjected to partial enzymatic digestionwith trypsin, cholesterol esterase, and neuraminidase according to themethod of Bhakdi^(50,66).

It was then dialysed into 0.05 M borate buffer, pH 8.0, and 10 μg totalprotein in 100 μl volumes were dispensed into the wells of CostarDNA-bind N-oxysuccinimide surface amine binding stripwell plates, andincubated at room temperature for 1 h. All wells were then washed threetimes with 200 μl volumes of PBS before blocking by incubation for 1 hat room temperature with 100 μl per well of 0.2 m Tris pH 8.0. The wellswere then washed again 3 times with 200 μl volumes of PBS and finallyemptied before adding to each one 100 μl volumes of the followingreagents. For control uninhibited maximal binding: a standard dose ofradiolabelled CRP³¹ in 0.01M Tris buffered 0.14M NaCl/0.002M CaCl₂ at pH8.0 containing 1% w/v BSA (TCB buffer). For background, non-specific,non-calcium dependent, binding in the presence of EDTA: the same dose ofradiolabelled CRP in 0.01M Tris buffered 0.14M NaCl at pH 8.0 containing10 mM EDTA, 1% w/v BSA (TEB buffer). For testing of inhibitors: thestandard dose of radiolabelled CRP that had been pre-incubated in TCB atroom temperature for 15 min with the various concentrations of testcompounds shown in the Table. The plates were then sealed and incubatedovernight at 37° C. in a sealed moist chamber, before 3 final washeswith 200 μl per well volumes of TCB or TEB, followed by air drying for 1h at room temperature, and then counting bound radiolabelled CRP.

The compounds tested in the experiment shown here were phosphocholine,which is known to be the best natural ligand for CRP,phosphoethanolamine, to which CRP is also well know to bind, andphosphoserine, a related phosphorylated molecule the binding to which ofCRP has not previously been reported. These were compared with hexadecylphosphocholine, the candidate inhibitor of CRP binding for use as a drugin vivo according to the present invention.

Results

The capacity of the compounds tested to inhibit binding of CRP to enzymemodified low density lipoprotein is expressed as the percentage by whichCRP binding was reduced compared to binding in the absence of anyinhibitor (Table 1). All binding was inhibited by EDTA, confirming thespecific, calcium dependent, nature of the interaction. Binding tocontrol wells without the specific E-LDL ligand was at the samebackground level as seen with complete inhibition by EDTA.Phosphoethanolamine, a well known ligand of SAP⁶⁷, produced inhibitionat high concentration, but only modest effects when diluted.Phosphocholine, as expected, was the most potent inhibitor of CRPbinding, but hexadecyl phosphocholine was also effective, with IC₅₀value in the micromolar range and as active or more thanphosphoethanolamine. Hexadecyl phosphocholine is thus suitable forfurther testing as an drug to inhibit pathogenetic effects of CRP invivo, according to the present invention.

Experiment Mediation of Tissue Damage by CRP and Complement in AcuteMyocardial Infarction.

This experiment demonstrates that parenteral injection of human CRPmarkedly enhances tissue damage, via a complement dependent mechanism,in experimental acute myocardial infarction produced by coronary arteryligation.

Materials and Methods

Protein Reagents and Assays. Human CRP human serum amyloid P component(SAP)⁶⁸, rat CRP⁶⁹, rat C3⁶⁹ and cobra (Naja naja) venom factor⁷⁰ wereisolated, purified and/or assayed precisely as previously described. Thepreparations of human CRP and SAP, and of cobra venom factor used invivo were all >99% pure.

Induction and Measurement of Myocardial Infarction. Female Wistar ratsbred at ICSM, Hammersmith campus were used at age 9-10 wk and weighed230-280 g. Within each experiment the whole body and heart weights werethe same in control and experimental groups. All animals were carefullyexamined and were healthy before being fasted overnight prior tosurgery. General anesthesia was induced with inhaled isoflurane andsupplemental oxygen was provided at 1.0 liter/min. The chest was openedthrough the sixth intercostal space, anesthesia was discontinued butsupplemental oxygen continued at 0.5 liter/min, and the pericardium wasopened to allow the heart to be lifted out of the thorax. The leftanterior descending coronary artery was immediately ligated at aconstant distance just below the atrium with a 5/0 silk suture, theheart was replaced and the chest closed whilst active ventilation with100% oxygen was continued. The duration of pneumothorax was about 30 s.Resuscitation was completed by gentle chest massage with the rat lyingsupine and there was usually rapid recovery of consciousness and of therighting reflex. Buprenorphine 0.03 mg/kg was given immediately forpost-operative analgesia and repeated 12 hourly. Development ofmyocardial infarction was confirmed by early post-operative 4 leadelectrocardiogram showing marked ST segment elevation in leads I and aVLas well as various arrhythmias.

Intra-operative and immediate post-operative mortality was up to 20%,but thereafter there were few deaths prior to day 5 when the rats werekilled for measurement of infarct size. Deep anesthesia was induced withisoflurane and the hearts were excised while still beating in sinusrhythm and immediately arrested in diastole by immersion in 10 ml of 30mM KCl. They were then cleaned of any extraneous adherent tissue andweighed before being briefly chilled at −20° C. to produce sufficientrigidity to facilitate cutting into defined sections. Each heart was cutinto four slices of equal thickness perpendicular to the course of theleft coronary artery, starting from the apex of the heart and ending atthe position of the ligature around that artery. These slices weredesignated A, B, C, and D starting from the apex, and each was washedwith pure water and then immersed in 0.02 M phosphate buffer pH 7.4containing 0.5 mg/ml of nitroblue tetrazolium (NBT) (Sigma) at 37° C.for 30 min. NBT stains viable but not infarcted myocardium. Afterstaining with NBT the slices were washed briefly in cold water beforefixation in 10% buffered formalin for 48 h, and the proximal cut surfaceof each slice was then imaged under standard conditions with a highresolution digital camera. The captured images were coded and analyzed‘blind’ without knowledge by the operators of the treatment received bythe rat in question. Lines were drawn around the total area of theslice, the NBT negative infarct zone, and any parts of the image to beexcluded from the estimation including valves, chordae tendinae, shadowsand edges, and the area of the infarct was determined as a percentage ofthe total area. The infarcts were confined to slices B, C and D, and themean percentage in these three was taken as the infarct size in thatheart. Statistical significance of differences between infarct sizes indifferent treatment groups within each experiment were sought by one wayANOVA and by Bonferroni t-tests.

In Vivo Treatments. Complement depletion was induced in vivo by a singlei.p. injection of cobra venom factor at 250 U/kg. Human CRP or SAP wereinjected i.p. at 40 mg/kg 1 h after coronary artery ligation and then at24 h intervals until the rats were killed on day 5. The proteins weredissolved in 10 mM Tris, 140 mM NaCl, pH 8.0, also containing 2 mM CaCl₂in the case of human CRP, and the control rats received injections ofthe buffer alone at the same intervals. In one experiment rats werekilled at 24, 48 and 72 h after coronary artery ligation. For analysisof the effect of cobra venom factor on plasma C3, the usual dose wasgiven and normal, unoperated rats were bled from the tail at intervalsup to 6 d. For measurement of plasma clearance of human CRP and SAP,normal unoperated rats received a single 40 mg/kg dose of each proteinand were bled at intervals up to 72 h later. Plasma clearance of humanCRP was also studied in the same protocol in normal unoperated rats thathad received cobra venom factor 24 h previously. These experiments couldnot be done in rats which had undergone coronary artery ligation becausethey do not survive the anesthesia required for bleeding, but there isno reason to suppose that the results in them would be significantlydifferent.

Immunohistochemical Analysis. The formalin fixed heart slices wereembedded in wax and sectioned at 2 μm thickness for routine hematoxylinand eosin staining and for immunohistochemical staining. Optimaldilutions of the IgG fractions of monospecific goat anti-human CRP,rabbit anti-rat CRP and sheep anti-rat C3 antisera, raised byimmunization with the respective isolated pure antigens, were used asprimary antibodies after blocking the sections by incubation in 20%vol/vol normal serum of the species of origin of the secondary antibody.In all cases endogenous peroxidase activity was blocked by incubationwith H₂O₂ before testing. Secondary antibodies were optimal dilutions ofgoat anti-rabbit IgG or rabbit anti-goat IgG (both from Dako), whichcross-reacts with sheep IgG. Finally rabbit or goatperoxidase-anti-peroxidase complexes (Dako) were used followed byvisualization with diaminobenzidine. All positive staining wascompletely abolished when the primary antibodies had been absorbedbefore use with an excess of the respective specific antigen. In thecase of rat C3, which was not available in pure form, parallelabsorptions of the sheep anti-rat C3 antibody were performed with normalrat serum and with C3 depleted serum from a cobra venom treated rat. Thelatter did not affect anti-C3 staining whilst C3 sufficient rat serumabolished it completely. In order to confirm that the antigens underinvestigation were not affected by formalin fixation and tissueprocessing, sample hearts were snap frozen without fixation and 5 μmcryostat sections were cut for immunoperoxidase staining. For allantigens, the appearances were identical to the fixed sections, exceptfor poorer preservation of histological detail as expected. Frozensections of a heart from a human CRP-treated rat killed at day 3 werealso stained for human CRP by indirect immunofluorescence, and thenuclei counterstained with ethidium bromide, in order to visualizebinding of CRP to cell surfaces.

Results

Infarct Size is Increased by Human CRP. We have previously shown thatrat CRP does not activate rat complement whereas human CRP does so veryefficiently⁶⁹. The rat model therefore permits specific analysis of thecomplement-dependent effect of human CRP on infarct size in vivo. Ratswhich received 40 mg/kg of isolated pure human CRP by i.p. injection 1 hafter coronary artery ligation rapidly became clinically less well thanbuffer-treated controls, and some died during the next 3 d whilstreceiving further daily injections of the same dose of human CRP. Incontrast rats which had not been operated on but just received the samedoses of human CRP showed absolutely no ill effects. Injections of humanSAP, the pentraxin protein very closely related to CRP¹, had no adverseclinical effects in either normal or coronary artery ligated rats. Whenall surviving animals were killed on day 5 after coronary arteryligation, the infarcts in those receiving human CRP were about 40%larger than in control rats treated either with buffer or human SAP(Table 2). The plasma clearance of human CRP and SAP following singlei.p. injections in control, non-operated rats, and the clearance ofhuman CRP in decomplemented rats, are shown in FIG. 1. Each datasetrepresents values in a single animal. □, human CRP in untreated rats;human CRP in rats decomplemented by i.p. injection of 250 U/kg of cobravenom factor 24 h beforehand; , human SAP in untreated rats.

The clearance of human CRP was not affected by complement depletion. Thepeak values for human CRP were comparable to massive acute phaseresponses in man², and those for human SAP were much higher than everseen in man⁷¹. Rats which have undergone coronary artery ligation do notsurvive the anesthesia required for bleeding and were therefore not bledbefore they were killed on day 5. However when these animals were killed24 h after the last of 5 daily injections, the serum concentrations ofhuman CRP were still in the range 13-53 mg/liter, typical of a moderateclinical acute phase response in man. In animals that had received humanSAP, the serum concentrations of this protein were between 45 and 175mg/liter. Values for rat CRP in serum at the time of exsanguination 5 dafter coronary artery ligation were 215-525 mg/liter, which is withinthe normal range for this species⁶⁹.

Complement Dependence of the Enhancement of Infarct Size by Human CRP.Administration of cobra venom factor in vivo rapidly produces profoundand sustained depletion of C3⁷⁰, with no active C3 remaining in thecirculation at 6 h. Traces of C3 antigen detectable thereafter areinactive cleavage fragments⁷². With the dose of cobra venom factor usedhere, 250 U/kg⁷⁰, active C3 starts to reappear in the circulation afterabout 4 d and is within the normal range by day 5 or 6. When rats hadbeen decomplemented by in vivo administration of cobra venom factor 24 hbefore coronary artery ligation, their infarcts at 5 d were about 60%smaller than in control untreated animals (Table 3). Complementsufficient rats injected daily with human CRP developed, as before,infarcts approximately 40% larger than those in control untreatedanimals. However, injection of human CRP had no effect at all on thereduced infarct size in decomplemented rats (Table 3). The damagingeffect of human CRP in this model is thus absolutely complementdependent.

Deposition of CRP and Complement in Rat Myocardial Infarcts. FIG. 2shows the results of immunohistochemical staining of rat myocardialinfarcts on day 5. a, hematoxylin and eosin stain showing infarctedmyocardial cells and adjacent dense mononuclear cell infiltrate. b,immunostain with anti-human CRP with uptake localized to the infarctedarea and both diffuse and focal patterns of immunoreactivity; many ofthe adjacent infiltrating macrophages are also immunoreactive. C,immunostain with anti-human CRP preabsorbed with isolated pure humanCRP, showing complete absence of any staining and confirming theimmunospecificity for human CRP of the pattern observed in b. d,immunostain with anti-rat C3 with uptake confined, in contrast to theanti-human CRP (b), to focal structures, possibly nuclear ghosts, withinthe infarct. e, Immunostain with anti-rat C3 preabsorbed with whole ratserum, showing complete absence of any staining. Absorption of theanti-rat C3 antibody with C3 depleted rat serum did not affect thestaining pattern, confirming its immunospecificity for rat C3. Originalmagnifications, ×40. Human CRP and rat C3 (FIG. 2), and rat CRP (notshown), were all deposited in the infarcted myocardium. On day 5 humanand rat CRP were present homogeneously throughout the infarcted muscleand also in a more intense, speckled pattern in multiple foci thatoccasionally coincided with hematoxyphil nuclear remnants. These focimay be nuclear ghosts from which chromatin has been cleared, butretaining the small nuclear ribonucleoprotein particles to which humanCRP binds avidly^(8,9) and possibly other CRP ligands. Rat C3 waspresent predominantly in the same speckled foci, with no diffuseimmunoreactivity on the infarcted muscle cells, in contrast to thedistribution of CRP. Immunofluorescence staining of sections of unfixed,snap frozen myocardial tissue taken 72 h after coronary ligation,clearly demonstrated the presence of CRP on the surface of damagedmyocardial cells in and around the infarct, as well as the samedistribution seen in fixed sections stained by the immunoperoxidasemethod. Staining for human CRP was always more intense than for rat CRP,although it is not clear whether this represents greater abundance ofthe human protein in the tissue sections, or just greater sensitivity ofthe respective immunostaining procedure. Numerous mononuclear cells inthe dense peri-infarct infiltrate also stained strongly for human CRP(FIG. 2) but not for rat CRP (not shown) or rat C3 (FIG. 2).

Discussion

CRP has been very stably conserved in evolution^(1,73,74) and nostructural polymorphism or deficiency of CRP in man has yet beenreported, suggesting that this protein has important normal functionsthat contribute to survival. In experimental models CRP is protectiveagainst pneumococcal infection and may contribute to innate immunity toother micro-organisms to which it binds¹³. CRP also probably plays animportant role in scavenging autologous ligands and preventingdevelopment of autoimmunity, by analogy with this role of SAP inrelation to chromatin that we have recently demonstrated⁷⁷.

The present results unequivocally demonstrate, in a robust experimentalmodel, that human CRP markedly enhances the extent of myocardial damageproduced by ischemic injury. Although the time-concentration profile ofhuman CRP produced by daily CRP injections was not the same as themonophasic acute phase response that follows uncomplicated naturallyoccurring myocardial infarction in man, it was comparable to thepersistent, high and fluctuating CRP pattern typically found in patientswith post-infarct complications⁵², and was therefore not‘unphysiological’ Endogenous rat CRP, as well as the injected human CRP,was deposited in the infarct in vivo, but rat CRP does not activate ratcomplement⁶⁹. In contrast human CRP is a potent activator of ratcomplement⁶⁹, and the enhancement of infarct size caused byadministration of human CRP is completely abrogated by in vivocomplement depletion. Human CRP production is always greatly increasedfollowing acute myocardial infarction, CRP is always deposited in humanmyocardial infarcts, and early and late clinical outcomes aresignificantly associated with peak post-infarction plasma levels of CRP.It is therefore very likely that CRP contributes importantly to theextent of damage in human acute myocardial infarction and, based on ourresults here, it probably does so via complement activation. Althoughcomplement activation by CRP is not efficient in generating the terminallytic complement complex, it very effectively cleaves C3, the criticalstep for opsonisation by C3b and liberation of the C3a anaphylatoxin.

These findings have important therapeutic implications, suggesting thata drug capable of inhibiting the binding of human CRP to its targetligands in vivo, and thereby preventing it from activating complement,should reduce infarct size with corresponding clinical benefit.Furthermore, increased CRP production is a feature of the non-specificacute phase response to a very wide range of traumatic, infectious,inflammatory and neoplastic tissue damaging conditions. In all of these,including disorders as diverse as burns, surgical trauma, rheumatoidarthritis, sepsis and invasive neoplasia, there are non-irremediablydamaged cells which, by analogy with myocardial infarction, are likelyto be targeted by the opsonic and pro-inflammatory actions of CRP andcomplement. Specific inhibition of CRP binding in vivo might thus beexpected to be of wide clinical benefit, and suitable compounds arecurrently being sought, supported by our recent description of the highresolution 3D structure of the physiological CRP-ligand complex

TABLE 1 Percent Inhibition by Various Compounds of Binding ofRadiolabelled CRP to Immobilized Enzyme Modified Low Density LipoproteinMolarity Compound 10 mM 1 mM 100 μM 10 μM 1 μM None 0 0 0 0Phosphoethanolamine 9 90 39 0 0 Phosphocholine 9 94 93 46 0 Hexadecylphosphocholine 9 73 76 0 0 Phosphoserine 3 0 0 0 0

TABLE 2 Human CRP Increases Myocardial Infarct Size in Rats InfarctBonferroni No. Day 5 size t-test Treatment treated survivors mean (SD) %vs controls Experiment 1 Buffer only 5 5 14.5 (1.1) Human CRP 5 4 21.2(2.0) P = 0.0007 Experiment 2 Buffer only 3 3 12.8 (0.9) Human SAP 5 512.4 (1.9) Not significant Human CRP 5 3 17.6 (0.4) P = 0.0022 Human CRPor SAP were injected i.p. at 40 mg/kg

TABLE 3 Complement Dependence of the Enhancement of Infarct Size byHuman CRP Infarct Bonferroni No. Day 5 size t-test Treatment treatedsurvivors mean (SD) % vs controls Buffer only 8 8 14.3 (1.2) Human CRP 53 19.7 (1.6) P = 0.0020 Complement 5 5  6.5 (0.9) P = 0.0000 depletionComplement 6 6  6.9 (0.3) P = 0.0000 depletion + human CRP Human CRP at40 mg/kg was injected i.p. 1 h after coronary artery ligation and thenat 24 h intervals until all rats were killed on 5 d later; controlsreceived just buffer alone. Parallel groups had been decomplemented byi.p. injection of cobra venom factor at 250 U/kg 24 h before coronaryartery ligation.

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1. A method for the treatment or prevention of tissue damage in asubject having an inflammatory and/or tissue damaging condition, whichcomprises administering to the subject an effective amount of a compoundcapable of inhibiting the binding of C-reactive protein (CRP) to anautologous or extrinsic ligand thereof.
 2. A method according to claim1, wherein the inflammatory and/or tissue damaging condition comprisesatherosclerosis.
 3. A method according to claim 1, wherein theinflammatory and/or tissue damaging condition is selected from aninfection, an allergic complication of infection, an inflammatorydisease, ischemic or other necrosis, traumatic tissue damage andmalignant neoplasia.
 4. A method according to claim 3, wherein thecondition is an infection selected from a bacterial infection, a viralinfection, and a parasitic infection.
 5. A method according to claim 3,wherein the condition is an allergic complication of infection selectedfrom rheumatic fever, glomerulonephritis, and erythema nodosum leprosum.6. A method according to claim 3, wherein the condition is aninflammatory disease selected from Rheumatoid arthritis, Juvenilechronic (rheumatoid) arthritis, Ankylosing spondylitis, Psoriaticarthritis, Systemic vasculitis, Polymyalgia rheumatica, Reiter'sdisease, Crohn's disease and Familial Mediterranean fever.
 7. A methodaccording to claim 3, wherein the condition is tissue necrosis selectedfrom Myocardial infarction, Tumour embolization and Acute pancreatitis.8. A method according to claim 3, wherein the condition is traumaselected from elective surgery, burns, chemical injury, fractures andcompression injury.
 9. A method according to claim 3, wherein thecondition is malignant neoplasia selected from Lymphoma, Hodgkin'sdisease, Carcinoma and Sarcoma.
 10. A method for the treatment orprevention of tissue damage in a subject having an inflammatory and/ortissue damaging condition which comprises administering to the subjectan effective amount of a compound of general formula (I):

wherein X is H or an organic substituent group, and Y is N substitutedto form ammonium.
 11. A method according to claim 10, wherein X is H orC₁ to C₂₀ alkyl.
 12. A method according to claim 11, wherein X is C₁₂ toC₂₀ alkyl.
 13. A method according to claim 10, wherein Y is N—R₃, inwhich each R is independently selected from C₁ to C₅ alkyl.
 14. A methodaccording to claim 13, wherein each R is CH₃.
 15. A method for thetreatment or prevention of tissue damage in a subject having aninflammatory/tissue damaging condition, which comprises administering tothe subject an effective amount of a compound comprisinghexadecylphosphocholine.
 16. A method for the treatment or prevention ofatherosclerosis in a subject which comprises administering to thesubject an effective amount of a compound capable of inhibiting bindingof C-reactive protein (CRP) to an autologous or extrinsic ligandthereof.
 17. A method for the treatment or prevention of tissue damagein a subject having a myocardial infarction, which comprisesadministering to the subject an effective amount of a compound capableof inhibiting binding of C-reactive protein (CRP) to its autologous orextrinsic ligand thereof at or after the onset of the infarction.
 18. Amethod for the treatment or prevention of a thrombotic complication ofatherosclerosis in a subject which comprises administering to thesubject an effective amount of a compound capable of inhibiting bindingof C-reactive protein (CRP) to an autologous or extrinsic ligandthereof.
 19. A method according to claim 1, wherein the subject is ahuman subject.
 20. A method according to claim 16, wherein the compoundcapable of inhibiting the binding of CRP to an autologous or extrinsicligand thereof has the general formula (I):

wherein X is H or an organic substituent group, and Y is N substitutedto form ammonium.
 21. A method according to claim 20, wherein X is H orC₁ to C₂₀ alkyl.
 22. A method according to claim 21, wherein X is C₁₂ toC₂₀ alkyl.
 23. A method according to claim 20, wherein Y is N—R₃, inwhich each R is independently selected from C₁ to C₅ alkyl.
 24. A methodaccording to claim 23, wherein each R is CH₃.
 25. A method for thetreatment or prevention of tissue damage in a subject with myocardialinfarction, which comprises administering to the subject an effectiveamount of a compound comprising hexadecylphosphocholine.
 26. A methodfor selecting a pharmaceutical compound for treating or preventingtissue damage in a subject having an inflammatory and/or tissue damagingcondition, which comprises contacting C-reactive protein (CRP) with aligand thereof under conditions to permit CRP ligand binding, in thepresence of a test compound; and selecting the test compound as thepharmaceutical compound If the test compound inhibits binding of CRP tothe ligand.
 27. A method according to claim 26, wherein a firstcomponent comprising one of CRP or the ligand thereof is present as partof a solid phase, which is contacted with a second component comprisingthe other as part of a liquid phase; and the step of testing for CRPligand binding comprises detecting binding of the second component tothe solid phase.
 28. A method according to claim 27, wherein the solidphase comprises the first component attached to a solid support.
 29. Amethod according to claim 28, wherein the solid support comprises aparticulate support or a solid surface.
 30. A method according to claim29, wherein the solid support comprises a solid surface comprising aninterior surface of a container.
 31. A method according to claim 27,wherein the step of testing for CRP ligand binding further compriseswashing the solid phase to remove unbound material.
 32. A methodaccording to claim 27, wherein the second component is labelled with adetectable label.
 33. A method according to claim 32, wherein thedetectable label comprises a radiolabel, a fluorochrome or an enzyme.34. A method according to claim 27, wherein the binding of the secondcomponent to the solid phase is detected immunologically.
 35. A methodfor selecting a pharmaceutical compound for treating or preventingtissue damage from a plurality of test compounds, which comprisesproviding an array of reaction zones and a plurality of test compounds,and selecting the pharmaceutical compound by performing the method ofclaim 26 in each reaction zone.
 36. A method according to claim 35,wherein the array of reaction zones comprises an array of containers.37. A method according to claim 36, wherein the array of containerscomprises a microtitre plate well array.
 38. A process for theproduction of a pharmaceutical agent, which process comprises (i)identifying a pharmaceutical compound by selecting the compoundaccording to the method of claim 26; and (ii) producing a pharmaceuticalagent by providing the pharmaceutical compound or a pharmaceuticallyacceptable derivative thereof.
 39. The method of claim 1 wherein thecompound is an antibody or fragment thereof that specifically bindsC-reactive protein.
 40. The method of claim 1 wherein the compound is afragment of C-reactive protein or a fusion protein comprising a fragmentof C-reactive protein.
 41. The method of claim 39 wherein said antibodyspecifically binds the calcium-dependent binding site of C-reactiveprotein.
 42. A method according to claim 10, wherein the subject is ahuman subject.
 43. A method according to claim 15, wherein the subjectis a human subject.
 44. A method according to claim 16, wherein thesubject is a human subject.
 45. A method according to claim 17, whereinthe subject is a human subject.
 46. A method according to claim 1,wherein the subject is a human subject.
 47. A method according to claim17, wherein the compound capable of inhibiting the binding of CRP to anautologous or extrinsic ligand thereof has the general formula (I):

wherein X is H or an organic substituent, and Y is N substituted to formammonium.
 48. A method according to claim 18, wherein the compoundcapable of inhibiting the binding of CRP to an autologous or extrinsicligand thereof has the general formula (I):

wherein X is H or an organic substituent, and Y is N substituted to formammonium.